Drug Uptake Pathways of Multidrug Transporter AcrB Studied by Molecular Simulations and Site-Directed Mutagenesis Experiments

Multidrug
resistance has been a critical issue in current chemotherapy.
In Escherichia coli, a major efflux
pump responsible for the multidrug resistance contains a transporter
AcrB. Crystallographic studies and mutational assays of AcrB provided
much of structural and overall functional insights, which led to the
functionally rotating mechanism. However, the drug uptake pathways
are somewhat controversial because at least two possible pathways,
the vestibule and the cleft paths, were suggested. Here, combining
molecular simulations and site-directed mutagenesis experiments, we
addressed the uptake mechanism finding that the drug uptake pathways
can be significantly different depending on the properties of drugs.
First, in the computational free energy analysis of drug movements
along AcrB tunnels, we found a ligand-dependent drug uptake mechanism.
With the same molecular sizes, drugs that are both strongly hydrophobic
and lipophilic were preferentially taken in via the vestibule path,
while other drugs favored the cleft path. Second, direct simulations
realized totally about 3500 events of drug uptake by AcrB for a broad
range of drug property. These simulations confirmed the ligand-dependent
drug uptake and further suggested that a smaller drug favors the vestibule
path, while a larger one is taken in via the cleft path. Moreover,
the direct simulations identified an alternative uptake path which
is not visible in the crystal structure. Third, site-directed mutagenesis
of AcrB in E. coli verified that mutations
of residues located along the newly identified path significantly
reduced the efflux efficiency, supporting its relevance in in vivo function.